PROJECT SUMMARY Neurodevelopment and cognitive function are among the most important public health outcomes. Loss of functioning early in life due to toxins, such as metals, can have lifelong impacts. The prevention of neurodevelopmental disorders of fetal origin is impeded by the lack of objective tools for early detection of susceptible individuals. We will address the role of prenatal metal exposure on neurodevelopment, integrating this work with the overall goal of understanding how metal exposures impact placental cues, specifically release of extracellular vesicles (EVs), which facilitate and direct neurodevelopment. It has been shown that placental trophoblasts actively release EVs, i.e., nano-sized membrane-bound vesicles, in both the maternal and fetal circulation. EVs shuttle cargoes of bioactive molecules from trophoblasts to recipient cells, such as microRNAs (miRNAs) that prime maternal tissues to support fetal development. In the fetal circulation, EVs can cross the blood-brain barrier, particularly in prenatal life when the barrier around the fetal brain is still porous and permeable. Not only is the placenta a primary target of metal toxicities, data show that EV signaling is sensitive to environmental influences, including metals. No study to date has investigated whether exposures to metals during the prenatal period determine alterations of circulating EV signals that, in turn, may help to diagnose and predict alterations in neurodevelopment. Notably, many placental miRNAs are known to regulate neurodevelopment and have significant overrepresentation of Gene Ontology terms associated with neurogenesis. We propose that in utero metal exposure disrupt normal miRNA expression in placental tissue and their release in circulating EVs, thus altering neural stem cell proliferation, self-renewal and differentiation during fetal development. In turn, we propose that EV-packaged miRNAs in the maternal circulation in pregnancy reflect early biological settings of this fetal programming. We will conduct a coordinated series of human studies and in-vitro experiments to: 1) characterize the risk for maladaptive neurodevelopment from prenatal exposure to toxic metals and their mixtures; 2) identify EV-encapsulated miRNAs released by the placenta in response to metals by accessing this novel form of prenatal signaling through a maternal blood draw during pregnancy, cord blood, and placental biopsy at birth; and 3) conduct in-vitro experiments to assess whether EV-encapsulated miRNAs are released by trophoblasts after treatment with individual metals or their mixtures; and whether miRNAs in EVs released from metal-treated trophoblasts alter critical functions of neural stem cells. We leverage the PRogramming of Intergenerational Stress Mechanisms (PRISM) cohort with prospective assessment of behavioral disinhibition in N=470 children followed to age 48 months. While we propose underlying theoretical pathways, the primary goal is to search for biomarkers of early risk. However, findings may also advance our understanding of molecular pathways that influence neurodevelopmental risk, which will be critical for the development of preventative strategies and possible therapeutic interventions.